This proposal describes a five-year training program for the development of an academic research career in basic science. The principal investigator is a postdoctoral research fellow at The University of Iowa. Her long-term goal in this project is the establishment of an independent research career studying the cell biology of redox-signaling in health and disease. Her mentor is Dr. John Engelhardt, professor and Chair of the Department of Anatomy and Cell Biology. The research environment includes access to all equipment and supplies needed as well as an advisory committee of faculty members with expertise in areas of interest to the principal investigator. Intracellular redox-regulated signaling pathways are coordinated by the spatially controlled production of reactive oxygen species within various subcellular compartments. Despite the fact that it has been known for over three decades that superoxide is produced locally in the nucleus, its function and regulation at this site remain completely unknown. The broad, long-term objective of this project is to investigate how superoxide-producing enzymes at nuclear membranes are activated and regulated, and how they may be involved in regulating the transcription of injury response genes. Our preliminary data indicate that nuclear NADPH oxidase isoform 4 (NOX4) is functionally distinct from the NOX4 complex found elsewhere in the cell. Our first aim will investigate the subunit composition of the nuclear NOX4 complex and elucidate the topology and permeability of superoxide production by nuclear membranes. In aim 2, we hypothesize that NOX4 regulation in the nucleus is in part dependent on the localized availability of its substrate NADPH and that glucose-6-phosphate dehydrogenase in the nucleus is recruited to NOX4 under conditions of LPS stress (which we know elevates nuclear NOX4 activity). Aim 3 hypothesizes that nuclear superoxide plays a role in controlling the activity of several redox-regulated transcription factors, including NFkB and AP-1. The control of these transcription factors by nuclear superoxide signals will be studied using several transcription factor reporter assays. These studies provide the first investigation of physiological functions and regulation of nuclear superoxide. PUBLIC HEALTH RELEVANCE: LPS released by gram-negative bacterial infections can cause rapid clinical deterioration and shock, often requiring care in an ICU setting. This proposal seeks to understand how LPS leads to an increase in nuclear superoxide free radical production by liver cells. The proposed research will enhance our understanding of LPS-mediated injury and may lead to new therapeutic approaches for endotoxin-mediated sepsis.